【作者】 王莉；

【导师】 高红亮； 李平作；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 转谷氨酰胺酶（Transglutaminase，EC2.3.2.13，TGase）是一种广泛存在于哺乳动物、鱼类、植物、细菌、真菌和绿藻等生物中的一类酶。它可以催化蛋白质分子内及分子之间的共价交联聚合，因此能够直接改变蛋白质本身以及蛋白质所附着的细胞、组织、甚至器官的特性，与血液凝固、伤口愈合、表皮角质化、红细胞膜硬化等生物现象有关，并参与调节细胞生长、分化、增生等生物学功能。它能够提高或改变许多蛋白的成胶性能，在食品、生物医药等方面有着广泛的应用前景。 我们采用PCR技术克隆了轮枝链霉菌（Streptomyces mobaraensis）的转谷氨酰胺酶（MTG）基因，并将其装载入pBV220，pET22b，pET30a等高效表达质粒，转化相应的大肠杆菌宿主菌，研究其表达特性。根据酶蛋白表达量和酶活性对重组菌进行筛选，得到MTG的最适异源表达重组菌pET30a-MTG／BL21（DE3）。在37℃，1mM的IPTG诱导5小时的条件下，MTG表达量最大，达到0.44U／ml发酵液。用商业TGase通过皮下注射兔制备得到抗血清，并对重组MTG进行了Dot blotting和Westen blotting鉴定。 Ni离子金属螯合层析纯化MTG的研究表明，以50mM的咪唑缓冲溶液（20mMNa₃PO₄，0.5MNaCl，pH7.4）洗脱，MTG的回收率较高，以总蛋白计回收率可达到69.2％。每升发酵液可制备约13mg纯MTG，经化学方法测定其酶活约为15.1U／mg蛋白，是目前所报道的基因工程MTG中最高的。酶的稳定性酶学性质研究表明：MTG的最适保存缓冲液为：25mMTris-HCl（pH6.0），内含0.5mM EDTA，10mM DTT，-80℃保存。重组MTG的最适作用pH6.5，最适作用温度40℃。用SDS-PAGE对其交联BSA蛋白的效果进行的功能鉴定表明：重组MTG与天然或商业化MTG具有同样的蛋白交联活性。更多还原

【Abstract】 Transglutaminases (protein-glutamine: amine y-glutamyltransferase, EC 2.3.2.13, TGase) widely distributed in most tissues, body fluids including the liver, hair follicles, epidermis, prostate, and blood. TGase is thought to be involved in diverse biological functions, such as maintenance of gross forms of structures and limited degrees of extensibility. TGase is also capable to enhance or change a wide variety of protein’s binding properties, which has extensive potential applications in food industry and medical treatment.TGase gene from Streptoverticillium mobaraense (MTG )is cloned by PCR method. The sequence of MTG is the same as the literature. The expression vectors pET30a, pET22b, and pBV220 were used for the heterologous expression of MTG in E. colt. The highest expression level of MTG was obtained in the E.coli BL21 (DE3)/pET30a-MTG with a yield of 0.44 U/ml fermentation medium after 5 hours induction of ImM IPTG at 37℃. The recombinant MTG was confirmed by dot blotting and Western blotting. Investigation on the purification of MTG by Ni-affinity chromatography showed that 13mg purified MTG could be obtained from 1 liter fermentation medium with 69.2% recovery yield based on the the total protein. The specific activity of the purified MTG reached to 15.1U/mg MTG, which is the highest level among the reported genetic engineered strains. Experimental results on the stability and properties of MTG showed that under -80 癈 the optimized storage buffer, for the recombinant MTG is 25mM Tris-HCl (pH6.0) containing 0.5mM EDTA and lOmM DTT; the suitable pH and temperature were 6.5 and 40℃ respectively. The biological activity of recombinant MTG was verified by BSA conjunction reaction that was assayed by SDS-PAGE electrophoresis. The results showed that the recombinat MTG possessed the same activityas the native or commercialized MTG.更多还原